In the lab, autopsies of research animals have helped researchers make great
strides in understanding the various pathways and biological processes that
take place in mammalian cell biology. But that’s after the animal has been
euthanized and those biological processes have ceased.

Now, intravital imaging is allowing researchers to capture images of those
same biological processes in live animals. It’s providing answers about how
cells behave in resting tissue, how they interact with one another, exchange
information, respond to pathological stimuli, and mediate functions. In
addition, the technique is providing insight into disease processes that
take place in cells by defining the impact of specific disturbances in
real-time, allowing researchers to test concepts gathered in vitro while
providing instructive observations not readily captured by the histological
evaluation of tissue.

Now, intravital imaging is allowing researchers to capture images of those same biological processes in live animals. Source: John Lewis

At the Albert Einstein College of Medicine, Jeffrey Pollard is the director of the Center for the Study of Reproductive Biology and Women's Health and deputy director of the Albert Einstein Cancer Center. Source: John Pollard

At the Department of Oncology in the University of Alberta, Canada, associate professor John Lewis is studying the tumor microenvironment to functionally elucidate the molecular switches of tumorigenesis. Source: John Lewis

Claudio Vinegoni is an instructor at Harvard Medical School and also director of the In Vivo Microscopy Program in the Center for Systems Biology at Massachusetts General Hospital. Source: Claudio Vinegoni

Mice with Breast Cancer

At the Albert Einstein College of Medicine, Jeffrey Pollard is the director of
the Center for the Study of Reproductive Biology and Women's Health and
deputy director of the Albert Einstein Cancer Center. One of his goals is to
develop intravital imaging technologies that will make imaging-based cell
collection and gene expression analysis of tissue cells routine.

“The use of high-resolution imaging as a front end in the collection and
interpretation of DNA microarray data is a unique combination of advanced
technologies and potentially a vast improvement over existing methods of
immunocytochemistry and laser capture microdissection as front ends for DNA
chip analyses,” says Pollard.

One main advantage of intravital imaging is the ability to observe
cell-to-cell interactions, thus proving they occur in vivo. Using
photoswitchable fluorescent proteins such as the green-to-red transitioning
Dendra2, researchers can track individual cells as they travel through blood
vessels within the body.

But there are limitations. For one thing, the technology is reliant upon
fluorescently labeled cells. In addition, even using infrared lazers,
researchers can only image to a depth of 300-400 µm. And it’s still quite
expensive. In his recent study published in Nature Protocols(1),
the two-laser multiphoton microscope used by his team cost around $1
million.

In that article, Pollard and colleagues used the microscope for multichannel
intravital fluorescence imaging of a mouse model of breast cancer. The
instrument extended the wavelength range for excitation, thereby expanding
the number of simultaneously usable fluorophores, and markedly increased
single-to-noise via overclocking of detection. Overall, Pollard notes that
the procedure can be completed in less than 24 hours.

Roosting Chickens

At the Department of Oncology in the University of Alberta, Canada, associate
professor John Lewis is studying the tumor microenvironment to functionally
elucidate the molecular switches of tumorigenesis. Namely, he’s looking at
tumor neoangiogenesis and the acquisition of tumor cell motility. He is also
investigating novel nanoparticles that are being developed for the early
detection of prostate cancer, drug delivery, and the in vivo study of tumor
cell invasion and metastasis. All of these projects are facilitated by
long-term time-lapse intravital imaging of human cancer progression.

Most intravital imaging studies have been in mice, which Lewis believes limits
the data that are being collected. His solution is chicken embryos. “You
can generate embryos faster and in greater quantity than you can mice. You
can also see what is going on for days in chicken embryos compared to 6-8
hours in mice. The data from the embryos allows for more confidence
statistically,” says Lewis.

In an article in the Journal of Visualized Experiments (2), Lewis
demonstrated that chicken embryos can be a useful model in assessing the
vascular dynamics and the pharmacokinetics of xenografted human tumors. “The
structure and position of the chorioallantoic membrane (CAM) allows
high-quality image acquisition and accommodates many kinds of cancer
xenografts without invasive surgical procedures. Moreover, cancer tumor
xenografts implanted into the chorioallantoic membrane become vascularized
within seven days, offering a rapid, inexpensive and semi-high-throughput
means to assess the accumulation of nanoparticles in tumor tissue,” says
Lewis.

Lewis also noted that the cancer xenografts implanted in the CAM of the
chicken embryos were “accessible to the high-resolution optics of an upright
epifluorescence or confocal microscope; contextual and temporal information
regarding nanoparticle uptake in the tumor vasculature can be readily
obtained.”

In the end, the use of chicken embryos and other models might increase with
the continued interest in intravital imaging. The cancer xenografts tend to
grow laterally across the CAM, which results in tumors that are large but
less than 200 µm in depth. At this depth, a standard epifluorescence
microscope can image the entire tumor. “In contrast, tumors implanted in
either superficial or orthotopic sites within the mouse proliferate in three
dimensions, making it difficult to accurately localize nanoparticles deep
within these tumors by non-invasive techniques,” says Lewis.

Rabbit Arteries

Claudio Vinegoni is an instructor at Harvard Medical School and also director
of the In Vivo Microscopy Program in the Center for Systems Biology at
Massachusetts General Hospital. His research focuses on developing novel in
vivo imaging methods. His work in fluorescence molecular tomography has
increased the resolution possible for 3-D imaging in living mice.

Currently, he’s developing novel optical mesoscopic molecular imaging
techniques to generate in vivo 3-D data in optically diffusive
non-transparent living organisms up to a few millimeters in size such as Drosophila
melanogaster and zebrafish. These techniques provide both in vivo
anatomical and functional imaging. Other research activities in his lab
involve in vivo near infrared fluorescence imaging of protease activity in
rabbit models of atherosclerosis, the development of novel fiber-based
imaging systems and combined optical and opto-acoustic multispectral
tomographic imaging for in-vivo imaging applications.

“The great advantage of intravital imaging is that it allows you to have
direct access to mouse models and is a well-established technique. You can
do it for hours, days, and weeks,” says Vinegoni. “You don't have to cut
open the mouse repeatedly to see what is going on."

However, some organs are difficult to get a look at. "Imaging the heart
is a challenge. It beats a lot. The average mouse has a heartbeat of 350
beats per minute. It makes imaging difficult," says Vinegoni.

Despite these challenges, intravital imaging is providing researchers with a
window into the cellular processes in living animals and continuing to grow
in popularity, especially as it becomes more affordable.